Synthesis of Peptide–Adenine Conjugates as a New Tool for Monitoring Protease Activity

Article abstract of DOI:10.1002/ejoc.201801490

We took advantage of the powerful adenine SERS (Surface Enhanced Raman Spectroscopy) probe to design peptide–adenine conjugates as candidates for use as serine protease substrates. Whereas the direct introduction of the peptide sequence on the adenine exocyclic N6 amine gave an imidazopurinone derivative, the introduction of an aminoethyl linker between the adenine group and the peptide chain led to the expected candidate probes. These potential substrates were then evaluated for monitoring the hydrolytic activity of trypsin, used as a model protease, by HPLC and by SERS. We demonstrated that the Boc–VPR–adenine conjugate is a substrate of trypsin and constitutes a good starting point to design optimized substrates to monitor protease activity by SERS.

Full text of DOI:10.1002/ejoc.201801490

A Journal of
Accepted Article
Title: Synthesis of peptide-adenine conjugates as a new tool for the
protease activity monitoring
Authors: Nicolas Masurier, Feryel Soualmia, Pierre Sanchez, Valérie
Lefort, Mia Roué, Ludovic T. Maillard, Gilles Subra, Aline
Percot, and Chahrazade El Amri
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201801490
Link to VoR: http://dx.doi.org/10.1002/ejoc.201801490
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10.1002/ejoc.201801490
European Journal of Organic Chemistry
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Synthesis of peptide-adenine conjugates as a new tool for the
protease activity monitoring
Nicolas Masurier,* [a] Feryel Soualmia,[b] Pierre Sanchez,[a] Valérie Lefort,[b] Mia Roué,[b] Ludovic T.
Maillard,[a] Gilles Subra,[a] Aline Percot[c,d] and Chahrazade El Amri*[b]
Abstract: We took advantage of the powerful adenine SERS
(Surface Enhanced Raman Spectroscopy) probe to design peptide-
adenine conjugates as candidates for their use as serine protease
substrates. Whereas the direct introduction of the peptide sequence
on the adenine exocyclic N6 amine gave an imidazopurinone
derivative, the introduction of an aminoethyl linker between the
adenine group and the peptide chain led to the expected candidate
probes. These potential substrates were then evaluated for the
monitoring of the hydrolytic activity of trypsin, used as a model of
protease, by HPLC and by SERS. We demonstrated that Boc-VPR-
adenine conjugate was a substrate of trypsin and constitute a good
starting point to design optimized substrates to monitor the protease
activity by SERS.
Raman scattering (SERS) have been also considered. SERS is
a surface-sensitive technique involving signal enhancement with
molecular probe adsorption on rough metal or nanostructured
surfaces, allowing the detection of single molecules.[8] The use
of SERS in biomedical applications, in particular in enzymology,
is advantageous, since it is ideal for dilute media and SERS
circumvents fluorescence background.[8] Another striking
element is that it provides vibrational fingerprints of adsorbed
molecules even in mixtures.[9] Based on SERS principles,
several hydrolytic enzymes including lipases, esterases as well
as proteases were successfully investigated.[10–14]
On the other hand, adenine constitutes a very powerful SERS
probe, as little as 10-13 mol of adenyl residue can be detected
when brought in contact with the silver colloidal particles used
for SERS.[15–17] Consequently, we proposed to design,
synthesize and evaluate adenyl containing peptides as potential
substrates to monitor protease activity. First, the synthesis of
peptide probes was optimized. In a second step, these modified
peptides were evaluated by SERS for their ability to be
recognized and cleaved by trypsin as a serine protease model.
In parallel, a selected probe was evaluated using HPLC and
Introduction
Serine proteases are major actors in cell signalling and tissue
remodelling. Their deregulation is implicated in many diseases
including cancer, neurodegeneration, and inflammation.[1–3] Thus,
many efforts have been made to develop serine proteases
inhibitors.[4–7] In such a context, fluorescence spectrometry is a
lead technique to identify and characterize inhibitors.
Fluorescence assays are based on the use of peptide substrates
bearing fluorogenic groups that generate highly fluorescent
entities during hydrolytic digestion. However, during screening
for enzyme inhibition, quenching and/or autofluorescence could
hamper the proper selection and evaluation of inhibitors. To
circumvent this issue, other methods, generally based on
compared to
a known substrate incorporating an amino-
methylcoumarin (AMC) group.[18]
1st Strategy - Direct anchoring
Peptide 4
+
O
R
O
H
H
N
NH2
N
Serine
protease
R1
N
NH
N
N
N
H
R2
O
R
N
nN
x
N
N
H
N
H
1
3
chromatographic
techniques
(electrophoresis
or
liquid
chromatography) must be used. Alternate approaches, such as
surface enhanced Raman spectroscopy or surface-enhanced
Raman detection
and quantification ?
2nd Strategy - Anchoring via a linker
[a]
[b]
Prof. N. Masurier, P. Sanchez, Dr. L.T. Maillard, Prof. G. Subra
IBMM, Univ Montpellier, CNRS, ENSCM, Montpellier, France.
E-mail: nicolas.masurier@umontpellier.fr
NH2
NH2
N
Serine
protease
N
N
N
http://ibmmpeptide.com/
R
O
O
H
N
H
N
N
N
Dr. F. Soualmia, V. Lefort, M. Roué, Prof. C. El Amri
Sorbonne Universités, IBPS, UMR 8256, B2A, Biological Adaptation
and Ageing, Integrated Cellular Ageing and Inflammation, Molecular
& Functional Enzymology, 7 Quai St Bernard, F-75005 Paris,
France.
N
N
N
N
R1
H
H
Rn
5
R2
O
x
NH2
2
+
Peptide 4
E-mail: Chahrazade.el_amri@sorbonne-universite.fr
[c, d] Dr. A. Percot
Sorbonne Université, UMR 8233, MONARIS, c49, 4 Place, Jussieu,
Scheme 1. Design of the peptide-adenine conjugates as potential SERS
Paris F-75005, France. CNRS, IP2CT FR 2622, MONARIS, 4 Place
Jussieu, Paris F-75005, France.
probes.
Supporting information for this article is given via a link at the end of
the document.
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10.1002/ejoc.201801490
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Results and Discussion
Based on previously reported AMC-containing substrates,[7,13,18]
two peptide sequences were selected to design the targeted
probes: Succinyl-LLVY was chosen as
a substrate for
chymotrypsin-like proteases and Boc-VPR for trypsin-like
enzymes. Two strategies were envisioned to anchor the adenine
moiety (Scheme 1).
The first strategy consisted of the direct linking of the peptide
sequence on the adenine exocyclic N6 amine (compound 1).
With such a sequence, the native adenine 3 will be released
during enzymatic hydrolysis. The second strategy consisted of
the anchoring of the peptide onto the adenine N1 via a linker
(compound 2). With this framework, N1-aminoethyladenine
moiety 5 will be released during proteolysis.
Direct anchoring approach
Considering the first strategy (Scheme 2), adenine 3 was
coupled to Boc-Tyr-OH, using carbonyldiimidazole (CDI) as
activating agent and dry dimethylformamide (DMF) as the
solvent. N6-acyladenine derivative 6a was isolated in 22% yield.
After testing several additional coupling reactants (EDCI/HOBt,
HBTU, T3P), we found that polyphosphonic anhydride (T3P) in
DMF at 50°C for one night improved slightly the yield of
compound 6a (33% yield). Then, the Boc group was removed,
using a 1:1 trifluoroacetic acid/dichloromethane (TFA/DCM)
mixture. The mass spectrometry (MS) analysis of the resulting
crude mixture showed the formation of a main compound, which
possesses a molecular weight of 281 Da instead of 298 Da for
the expected Boc-deprotected adenine peptide conjugate 7. It
was identified as the tricyclic derivative 8a (Scheme 2). Such a
reactivity has already been observed by Chheda et al. with N6-
glycyladenine 6b,[19,20] which is converted into the tricyclic
derivative 8b in water after the loss of ammonia (scheme 3,
route i). These authors reported also that the tricyclic derivative
8b could further evolved to N-(6-purinyl)glycine 14b after ring
opening by a molecule of water. According to our experimental
conditions, the formation of a minority compound 9a (~15%) was
also detected in the crude with compound 8a (Scheme 2 and
ESI, Figures S1 and S2). Its molecular weight of 298 Da don't
match the acid 14a. Based on its fragmentation pattern (ESI,
Figures S3 and S4), compound 9a was identified as the amide
derivative (Schemes 2 and 3). As compound 9a was not
obtained after treatment of compound 8a with aqueous or
methanolic solution of ammonia (Scheme 3), the formation of 9a
could not result from the aminolysis of the lactame ring of 8a. A
possible mechanism to explain the formation of 9a is proposed
Scheme 3, route ii.
Scheme 2. Attempts to prepare the peptide-adenine conjugate using the direct
anchoring approach (First strategy).
As the synthesis of the peptide-adenine conjugate 1 was not
successful from N-acyladenine 6, we then envisioned its
synthesis by direct coupling adenine 3 and the protected
tetrapeptide Boc-LLVY-OH 4, which was prepared in solution,
using a classical Boc/Bn strategy (Scheme 4). Coupling of
peptide 4 with adenine 3 in DMF was studied, after activation of
4 by CDI or isobutylchloroformate (IBCF). However, even after
48h, only traces of conjugate 1 were detected by LC/MS
analysis. It is worth noting that this strategy could also yield to
tyrosine epimerization by oxazolone formation. Taken together,
these limitations lead us to envision the second strategy.
Scheme 3. Putative mechanisms for the formation of 8 and 9. In blue,
intermediates and structures proposed by Chheda et al..[19,20]
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BrCH2CH2NH2.HBr
Boc2O
K2CO3, DMF
NH2
NH2
NH2
1. TFA/DCM
BrCH2CH2NHBoc
N
N
N
N
N
N
N
18
2. BocAAOH,
EDCI, HOBt,
TEA, DMF
N
H
K2CO3, DMF
N
N
N
N
3
19
NHBoc
NH
O
NH2
N
N
NHBoc
R2
20a-d
N
N
20
Peptide
elongation
NH
O
O
NHBoc
N
H
21a-d
R2
n
R
21a : Boc-Leu-Leu-Val-Tyr(Bn)-NH-(CH2)2-Adenine
21b : Boc-Val-Pro-Arg(NO2)-NH-(CH2)2-Adenine
21c : Boc-Val-Pro-Orn(Cbz)-NH-(CH2)2-Adenine
21d : Boc-Val-Pro-Orn(Fmoc)-NH-(CH2)2-Adenine
Scheme 4. Synthesis of peptide Boc-LLVY-OH 4 and direct coupling between
adenine 3 and 4.
Scheme 5. Synthesis of the protected peptide-adenine conjugates 21a-d.
Anchoring via a linker
Firstly, N-Boc protected amino-ethyladenine 19 was prepared.
For that purpose, tert-butyl 2-bromo-ethylcarbamate 18 was
synthesized by protection of commercial 2-bromo-ethylamine
hydrobromide with tert-butylcarbonate, according to a slightly
modification of the procedure described by Narayanaswamy et
al.[21] Compound 18 was then used to alkylate adenine, using
potassium carbonate in DMF (Scheme 5). Compound 19 was
isolated in 46% yield after precipitation. The Boc group was then
removed in acidic conditions. The resulting deprotected
compound was then coupled with Boc-Tyr(Bn)-OH in DMF,
using EDCI/HOBt as coupling reagents to offer compound 20a in
20% yield. Chain elongation was then carried out using a
standard Boc strategy for peptide synthesis, in the same
conditions as previously, to lead protected compound 21a. The
Boc group of 21a was removed by treatment with a TFA/DCM
mixture (Scheme 6). The terminal amine was then condensed
with succinic anhydride. Finally, the benzyl group was removed
by hydrogenolysis yielding Suc-LLVY-adenine 22a, which was
purified by preparative HPLC.
Scheme 6. Synthesis of the peptide-adenine conjugates.
To access the second substrate which is based on a Boc-VPR
sequence, compound 19 was deprotected as previously and the
deprotected derivative was coupled with Boc-Arg(NO2)-OH. The
resulting compound 20b as well as all the other intermediates
during the peptide chain elongation revealed to be highly soluble
in water. Therefore, each coupling intermediate needed to be
purified by preparative HPLC, leading to a very poor final yield in
compound 21b (2% overall yield, starting from compound 19).
Nitro group was finally removed quantitatively by hydrogenolysis,
using Pd/C in ethanol to offer compound 22b (Scheme 6). To
improve the global yield, we envisioned to generate the
guanidine function of the arginine in the last step of the
synthesis. To achieve this goal, an ornithine derivative with a
Cbz-protected lateral chain was prepared (compound 20c,
Scheme 5).
Because of the higher lipophilicity of all the intermediates, by-
products could be easily removed by simple washing with
sodium hydrogenocarbonate solution. Compound 21c could be
recovered in 44% overall yield from 19. Cbz group of 21c was
then removed by hydrogenolysis, using Pd/C, with one
equivalent of hydrochloric acid. Surprisingly, this deprotection
step revealed to be slow and led finally to compound 22c and
several other unidentified derivatives, whatever the solvent
tested (EtOH, THF, AcOEt). Thus, we attempted to protect the
ornithine lateral chain with a Fmoc group. Fmoc-ornithine
derivative was prepared by protection of the commercially
available Boc-Orn-OH with Fmoc-O-succinimide in a mixture of
THF and water. Boc-Orn(Fmoc)OH derivative was obtained in
81% yield. After acidic deprotection of 19, the resulting product
was then coupled with Boc-Orn(Fmoc)OH, to offer compound
20d in 75% yield. Peptide chain elongation was then realized as
previously to obtain 21d in 46% yield. Fmoc group removal was
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realized with diethylamine in DMF. The deprotected derivative
22c was used in the next step without purification. Ornithine was
converted into arginine, using pyrrazole-carboxamidine
hydrochloride, as the guanidinylation agent, in DMF. Compound
22b was finally obtained after purification by preparative HPLC
in 12% overall yield, from compound 19.
Monitoring of the trypsin activity by SERS
The ability of compounds 22a and 22b to be recognized and
cleaved by trypsin was then evaluated by SERS, using the
Creighton silver colloid.[22] Experiments were performed in
triplicates. Several silver colloid batches were synthesized using
the classical Creighton’ method[22] and analyzed as described in
Soualmia et al..[13] Compound 22a revealed to be poorly soluble
in the aqueous medium at micromolar concentrations used for
the spectra registration. Thus, we focused our study on
compound 22b. The SERS spectrum of the peptide conjugate
Boc-VPR-Adenine 22b was compared to that of compound 5,
which illustrates the spectral fingerprint of the released amino-
ethyladenine moiety after the proteolysis.
Very low Raman signal was observed for 22b (Figure 1A, t= 0
min), whereas spectrum of compound 5 showed several more
intense Raman bands (spectrum identical to that obtained after
t= 40 min on Figure 1A). These bands (Table 1) could be
attributed to the adenine moiety, even if their positions shifted
slightly from canonical adenyl bands.[15,16] The overall fingerprint
confirmed a privilege-concerted (cooperative) C6-NH2/N7 binding
available for adenyl group with the intensity of the bands which
increased proportionally to the concentration of compound 5
(data not shown). Time-course of the tryptic hydrolysis of 22b
was then followed using a sampling method, with a substrate
concentration adjusted at 100 µM in order to have an optimum
SERS detection during the investigated time scale (Figure 2A).
Successive samples were collected over time from the reaction
medium and subjected to SERS measurement. Thus, registered
SERS spectra illustrated the composition of reaction media at a
given stage. The time course of compound 5 release is shown in
Figure 1A. At t= 0 min, the spectrum intensity is low. During
digestion process, the contribution of compound 5 in the spectra
increased over the time. Pattern spectra revealed the apparition
of adenyl characteristic bands (noted by asterisks in Figure 1A).
In addition, intensity of typical bands of the released adenyl
group (see above) increased over the time. Figure 1B presents
the increase of the intensity of the characteristic band at 1384
cm-1 as a function of time, these latter correlated to the increase
of compound 5 concentration. However, the evaluation of
kinetics parameters was not achieved due probably to a weaker
affinity of compound 5 compared to free adenine or improper
adsorption orientation onto silver colloid nanoparticles.
Figure 1. A: Time-course evolution of SERS spectrum of 100 µM 22b after
incubation with 200 nM of tryspin in Tris buffer 10 mM at pH 7.5. The final
percentage of DMSO was 2% for all samples. The spectra were normalized to
the water band at 3500 cm-1. Asterisks showed the adenyl characteristic bands.
B: Progression of SERS intensity 1384 cm-1 adenyl band over the time.
Monitoring of the trypsin activity by HPLC
The proteolytic cleavage of VPR-Adenine 22b by trypsin was
then studied using a HPLC technique. Firstly, 75 µM of 22b was
incubated at 37°C with trypsin in a TRIS-HCl buffer (100 mM, pH
8.0). The time-course of the reaction was followed by measuring
the apparition of Boc-VPR-OH at λ= 214 nm (Figure 2A and ESI
Figures S7 and S8). Rapidly, a new product was detected and
22b was totally hydrolyzed after around 1 hour of incubation.
LC-MS analysis (ESI+) of the final hydrolysis medium showed
that Boc-VPR-OH (RT= 1.11 min, [M+H]+= 471.2 Da) and
compound 5 (injection peak) were formed, demonstrating that
22b was a substrate of trypsin. The rates of hydrolysis of 22b
were then determined using 5 different concentrations of 22b
(37 to 150 µM). The rate data for 22b were plotted as double
reciprocal plots (Lineweaver-Burk plots of 1/rate vs 1/[substrate])
as shown in Figure 2B. From these plots, the values of Km (the
Michaelis constant) and Vmax (the maximal velocity of the
reaction) were determined. The catalytic constant was
Table 1. Main SERS observed bands and assignments of adenyl moieties.
Wavenumber (cm-1)
2928, 1014, 953
685-785
Assignments
DMSO
C6NH2 vibrational modes
C6NH2 vibrational modes
N7[Ag]n interaction
N7[Ag]n interaction
calculated from kcat
= Vmax/E0, where E0 is the enzyme
980-1045
concentration (200 nM) and finally the ratio kcat/Km was obtained.
These data are presented in Table 2. These data were
compared to those obtained with the classical Boc-VPR-AMC
substrate, using the same experimental procedure (see ESI,
1380-1423
1482
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European Journal of Organic Chemistry
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Figure S9). Based on the second-order rate constant (kcat/Km),
the value for compound 22b is about 6 times smaller than that
for Boc-VPR-AMC. The Km value for 22b is also smaller than
that for the AMC derivative. If Km reflects the binding affinity of
these two substrates to trypsin, 22b is a weaker substrate
relatively to Boc-VPR-AMC for trypsin, as already observed
using SERS.
trypsin, its cleavage product was detected by SERS. Kinetic
parameters were then determined using an HPLC technique and
compared to those obtained with Boc-VPR-AMC. Compound
22b revealed to be a weaker substrate for trypsin than the AMC
derivative (Km of 135.3 ± 2.7 µM compared to 11.1 ± 0.3 µM).
Ongoing studies especially on the linker chemistry may allow to
produce optimized substrates for SERS detection since adenine
constitutes one of the best SERS probe that could also be
expanded to other proteases.
Experimental Section
All reagents and solvents were from AlfaAesar and Acros and were used
without further purification. The following abbreviations were used: DCM,
dichloromethane;
DMF,
N-N’-dimethylformamide;
HPLC,
High
Performance Liquid Chromatography; LC/MS, Tandem Liquid
Chromatography/ Mass Spectroscopy; TFA, trifluoroacetic acid; THF,
Tetrahydrofuran. Other abbreviations used were those recommended by
the IUPAC-IUB Commission.[23] Chemical and enzymatic reactions were
monitored by HPLC using an analytical Chromolith Speed Rod RP-C18
185 Pm column (50 x 4.6 mm, 5 mm), at a flow rate of 3.0 mL/min, and
gradients of 100/0 to 0/100 eluents A/B for 5 min (eluent A= H2O/0.1%
TFA and B= CH3CN/0.1% TFA). Detection was performed at 214 nm
using a photodiode array detector. Retention times (RT) are reported in
min. 1H and 13C NMR spectra were recorded at room temperature in
deuterated solvents. Chemical shifts (δ) are expressed in parts per
million (ppm), relative to the resonance of CDCl3 = 7.26 ppm for 1H
(77.16 ppm for 13C), CD3OD = 3.31 ppm for 1H (49.00 ppm for 13C) or
DMSO d6 = 2.50 ppm for 1H (39.52 ppm for 13C). The following
abbreviations were used: s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), br (broad). LC-MS spectra (ESI+) were performed on a
Waters Alliance 2695, coupled with Waters MS ZQ and UV 2489
detectors. Detection was performed at λ = 214 nm, sample cone 20 V.
LC-MS-MS (ESI+) were performed on a Waters Alliance 2695, coupled
with Waters MS Quattro micro and UV 2488 detectors. Detection was
performed at 214 nm. Samples were analyzed using an analytical
Chromolith Speed Rod RP-C18 185 Pm column (25 x 4.6 mm, 2.5 mm);
solvent A, H2O/HCOOH 0.1%; solvent B, CH3CN/HCOOH 0.1%; gradient,
0% solvent B to 100% solvent B in solvent A in 2.5 min; flow rate, 3.0
Figure 2 A. Time course of tryptic hydrolysis of 22b (75 µM) by HPLC (λ= 214
nm). The amount of Boc-VPR-OH in nmol refers to the amount contained in 10
µL of the reaction mixture applied to the column at each time (average of 4
independent reactions). B. Double reciprocal (Lineweaver-Burk) plot of kinetic
data for the action of trypsin (200 nM) on 22b. Each point represents the
average of triplicate measurements.
mL/min.
RP-preparative HPLC purification was performed on a
Waters HPLC 4000 instrument, equipped with a UV detector 486 and a
C18 reversed-phase column. Standard conditions were eluent system A
(water/0.1% TFA), system B (acetonitrile/0.1% TFA). The crude sample
was dissolved in dimethylsulfoxyde (~100 mg in 3 mL). A flow rate of 50
mL/min and a gradient of (0–35)% B over 30 min were used, detection λ=
214 nm.
Table 2. Kinetic data.
Substrate
22b
Km (µM)
kcat (min-1)
72.6 ± 1.8
34.1 ± 0.6
kcat/Km (µM-1 min-1)
0.53 ± 0.02
General procedure for N6-acylation of adenine
135.3 ± 2.7
11.1 ± 0.3
To a stirred solution of Boc-Tyr-OH or Cbz-Tyr-OH (1.48 mmol) in
5 mL of DMF, 1036 µL of polyphosphonic anhydride solution at 50% in
DMF (1.62 mmol, 1.1 eq) and then 400 mg of adenine (2.96 mmol, 2 eq)
were added. The suspension was stirred at 50°C for one night. After
evaporation under vacuum, 50 mL of ethyl acetate were added and then
washed with water (25 mL) and then by an aqueous saturated NaHCO3
solution (2 x 25 mL). The organic layer was dried over Na2SO4, filtered,
and the solvent was concentrated under vacuum to offer compound 6 as
a white powder.
Boc-VPR-AMC
3.08 ± 0.13
Conclusions
We investigated the synthesis of adenine-peptide conjugates as
alternative to the AMC-serine proteases substrates. Whereas
the direct anchoring of the peptide chain onto the adenine
exocyclic N6 amine led only to the tricyclic compound 8, the
introduction of an aminoethyl linker between the adenine group
and the peptide chain led to the candidate probes. Then, we
bring experimental evidence that compound 22b may be
recognized although not very efficiently and could be cleaved by
N6-(N-Boc-tyrosyl)adenine 6a : White solid (196 mg, yield 33%);
1H NMR (DMSO d6, 300 MHz): δ ppm 1.81 (s, 9H), 3.47 (dd, 1H, J = 14.3
Hz, 8.4 Hz), 3.68 (dd, 1H, J = 14.3 Hz, 5.5 Hz), 5.20 (m, 1H), 7.20 (d, 2H,
J = 8.5 Hz), 7.63 (d, 2H, J = 8.5 Hz), 9.05 (s, 1H), 9.12 (s, 1H); HPLC,
RT= 1.09 min; MS (ESI+): m/z calc for C19H23N6O4 [M+H]+ 399.2, found
399.3
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N6-(N-Cbz-tyrosyl)adenine 6b : White solid (290 mg, yield 45%);
1H NMR (DMSO d6, 300 MHz): δ ppm 2.74 (d, 1H, J = 13.0 Hz), 3.03 (d,
1H, J = 13.0 Hz), 4.65 (bs, 1H), 4.97 (s, 2H), 6.66 (d, 2H, J = 7.0 Hz),
7.10-7.30 (m, 10H), 8.44 (s, 1H), 8.66 (s, 1H), 9.25 (bs, 1H); 13C NMR
(DMSO d6, 100 MHz): δ ppm 36.2, 57.0, 65.4, 114.9, 121.0, 126.8, 127.5,
127.6, 127.8, 128.3, 130.3, 136.8, 145.8, 151.2, 152.4, 155.9, 156.0,
172.7; HPLC, RT= 1.19 min; MS (ESI+): m/z calc for C22H21N6O4 [M+H]+
433.2, found 433.0
white powder (1.91 g, 46% yield). Spectral characteristics were in
agreement with the published data.[24]
Synthesis of Boc-Orn(Fmoc)-OH
Boc-Orn-OH (500 mg, 2.15 mmol) was dissolved in 16 mL of a mixture of
THF/H2O 1/1 v/v. Sodium bicarbonate (360 mg, 4.28 mmol, 2eq) was
then added, followed by Fmoc-OSu (730 mg, 2.15 mmol, 1 eq). The
reaction was stirred at room temperature for 2 hours. THF was removed
under vacuum and the aqueous layer was washed with Et2O (2 x 20 mL).
The aqueous layer was then acidified by 1N HCl and then extracted by
EtOAc (2 x 20 mL). The organic layers were dried over Na2SO4, filtered
and the solvent was concentrated under vacuum to offer compound as a
white powder (795 mg, 81% yield). 1H NMR (CDCl3, 300 MHz): δ ppm
1.23 (bs, 1H), 1.42 (s, 9H), 1.58-1.84 (m, 4H), 3.00-3.19 (m, 2H), 4.16-
4.22 (m, 4H), 5.22 (bs, 1H), 6.13 (bs, 1H), 7.27 (t, 2H, J = 6.7 Hz), 7.36 (t,
2H, J = 6.7 Hz), 7.55 (d, 2H, J = 6.7 Hz), 7.72 (d, 2H, J = 6.7 Hz); 13C
NMR (CDCl3, 75 MHz): δ ppm 25.5, 28.4, 29.8, 40.5, 47.3, 53.1, 66.8,
80.4, 120.1, 125.2, 127.2, 127.8, 141.4, 144.0, 155.9, 156.9, 176.0;
HPLC, RT= 1.85 min; MS (ESI+): m/z calcd for C25H31N2O6 [M+H]+ 455.2,
found 477.2 [M+Na]+, 355.2 (M+H-Boc]+.
Synthesis of compound 8a+9a
To compound 6a (0.13 mmol) were added 10 mL of a mixture of
TFA/DCM 50/50 v/v. The solution was stirred at room temperature for 1
hour. The solution was concentrated to dryness and co-evaporated
several times with acetonitrile to completely remove TFA. The residue
was dissolved in DCM (10 mL) and washed with a saturated NaHCO3
solution (2 x 10 mL) to offer compound 8a and compound 9a as a
mixture. Compound 8a : HPLC, Tr= 0.70 min; MS (ESI+): m/z calc for
C14H12N5O2 [M+H]+ 282.1, found 282.3; compound 9a : HPLC, RT= 0.66
min; MS (ESI+): m/z calc for C14H15N6O2 [M+H]+ 299.1, found 299.3
Synthesis of compound 4
General procedure for the synthesis of compounds 20
- Peptide coupling conditions: Boc-Tyr(Bn)OH.Ts or peptide 15 or 16
(0.75 mmol) was dissolved in DCM (10 mL) and basified with
triethylamine. HBTU (313 mg, 1.1 eq), Boc-Val-OH or Boc-Leu-OH (1.1
eq) were added. The solution was stirred at room temperature for 2 h and
then evaporated to dryness. The residue was dissolved in 25 mL of ethyl
acetate and washed with a saturated NaHCO3 solution (2 x 25 mL) and
then with brine (1 x 25 mL). The organic layer was dried over Na2SO4,
filtered, and the solvent was concentrated under vacuum.
A solution of compound 19 (200 mg, 0.72 mmol, 1 eq) in 10 mL of
DCM/TFA 1/1 v/v was stirred at room temperature for 1 h. After
evaporation to dryness under reduce pressure, the residue was dissolved
in DMF (5 mL), basified with triethylamine. 1-Hydroxybenzotriazole
monohydrate (121 mg, 0.79 mmol, 1.1 eq), EDCI (151 mg, 0.79 mmol,
1.1 eq) and Boc-Tyr(Bn)-OH or Boc-Arg(NO2)-OH or Boc-Orn(Cbz)-OH
or Boc-Orn(Fmoc)-OH (0.72 mmol, 1 eq) were added. The solution was
stirred at room temperature for 2 h and then evaporated to dryness. For
compound 20b, the crude was purified by preparative HPLC. For
compounds 20a, 20c and 20d, the residue was dissolved in 25 mL of
ethyl acetate and washed with a saturated NaHCO3 solution (2 x 25 mL)
and then with brine (1 x 25 mL). The organic layer was dried over
Na2SO4, filtered, and the solvent was concentrated under vacuum to offer
compound 20c and 20d.
Boc deprotection: removal of the Boc protecting group was carried out
using a solution of TFA/DCM 1/1 v/v at room temperature for 1h. The
solution was then evaporated to dryness under vacuum and the residue
was used in the next step without purification.
-
Benzyl deprotection: purified benzyl-protected derivative 17 was
dissolved in ethanol (20 mL). Two drops of 37% hydrochloric acid,
followed by 10% activated palladium on carbon were added. Hydrogen
gas was bubbled into the solution at 25°C until completion of the reaction
(HPLC monitoring). Then, the solution was filtered on a pad of celite.
Deprotected derivative 4 was obtained after evaporation to dryness
under vacuum, as a white powder (22% yield). HPLC, RT= 1.57 min; MS
(ESI+) m/z: calcd for C31H51N4O8 [M+H]+ 607.4, found 607.3.
Compound 20a : white powder (153 mg, 40 % yield). 1H NMR
(CDCl3, 300 MHz): δ ppm 1.38 (s, 9H), 2.90 (m, 2H), 3.48-3.65 (m, 2H),
4.27 (m, 1H), 4.97 (s, 2H), 5.18 (bs, 1H), 6.75 (bs, 1H), 6.81 (d, 2H, J =
8.6 Hz), 7.03 (d, 2H, J = 8.6 Hz), 7.25-7.38 (m, 5H), 7.64 (s, 1H), 8.06 (s,
1H); HPLC, RT= 1.29 min; MS (ESI+): m/z calcd for C25H35N8O5 [M+H]+
527.2, found 527.1.
Compound 20b : white powder (165 mg, 48 % yield). 1H NMR
(DMSO-d6, 300 MHz): δ ppm 1.36 (s, 9H), 3.09 (m, 4H), 3.49-3.76 (m,
5H), 4.26 (t, 2H, J = 5.7 Hz), 6.86 (d, 1H, J = 8.5 Hz), 7.92 (bs, 2H), 8.05
(t, 1H, J = 5.6 Hz), 8.28 (s, 1H), 8.36 (s, 1H), 8.62 (bs, 2H); 13C NMR
(DMSO-d6, 100 MHz): δ ppm 28.2, 28.9, 31.3, 38.4, 43.2, 51.9, 54.0,
78.1, 118.2, 143.1, 147.2, 149.0, 152.0, 155.3, 159.2, 172.4; HPLC, RT=
0.82 min; MS (ESI+): m/z calcd for C18H30N11O5 [M+H]+ 480.2, found
480.1.
Synthesis of tert-butyl 2-bromoethylcarbamate 18
To a stirred solution of 2-bromo-ethylamine hydrobromide (4 g, 20
mmol) in 40 mL of chloroform at 0°C, di-tert-butyl dicarbonate (4.29 g, 20
mmol, 1 eq) and then triethylamine (4.5 mL, 32 mmol, 1.7 eq) were
added. The reaction was allowed to stir at room temperature for 24 h.
Aminomethyl PS resin was then added (2 g, loading of 1.1 mmol.g-1, 0.1
eq) and the suspension was gently stirred for 1 h. After filtration, the
solution was washed with brine (2 x 40 mL), dried over Na2SO4. The
residue was purified by chromatography on silica, eluted with
nHex/AcOEt 4/1 v/v to offer a colourless liquid (4.33 g, 99% yield).
Spectral characteristics were in agreement with the published data.[21]
Compound 20c : white powder (352 mg, 93 % yield). 1H NMR
(DMSO-d6, 400 MHz): δ ppm 1.30-1.48 (m, 12H), 2.93 (m, 2H), 3.47 (m,
2H), 3.77 (m, 2H), 4.19 (t, 2H, J = 5.5 Hz), 5.00 (s, 2H), 6.80 (d, 1H, J =
7.9 Hz), 7.15 (bs, 2H), 7.19 (t, 1H, J = 5.9 Hz), 7.29-7.35 (m, 5H), 8.01 (s,
1H), 8.04 (t, 1H, J = 6.1 Hz), 8.13 (s, 1H); 13C NMR (DMSO-d6, 75 MHz):
δ ppm 26.5, 28.7, 29.6, 39.0, 40.7, 43.1, 54.6, 65.6, 78.5, 119.2, 128.2,
128.8, 137.7, 141.4, 144.0, 150.1, 152.8, 155.8, 156.4, 156.5, 172.9;
HPLC, RT= 1.29 min; MS (ESI+): m/z calcd for C25H35N8O5 [M+H]+ 527.2,
found 527.1.
Synthesis of 9-(2-N-Boc-aminoethyl)-9H-purin-6-ylamine 19
A
suspension of adenine 3 (2 g, 14.8 mmol), tert-butyl 2-
bromoethylcarbamate 18 (3.33 g, 14.8 mmol, 1 eq), potassium carbonate
(4.1 g, 29.7 mmol, 2 eq) in 60 mL of DMF was stirred at room
temperature for 3 days. The suspension was filtered and evaporated to
dryness under reduced pressure. The residue was triturated with water (5
mL), filtered and dry at 40°C under vacuum to offer compound 19 as a
Compound 20d : white powder (302 mg, 75 % yield). 1H NMR
(DMSO-d6, 300 MHz): δ ppm 1.31 (m, 2H), 1.36 (s, 9H), 1.40-1.57 (m,
2H), 2.91 (m, 2H), 3.50 (m, 2H), 3.72 (m, 1H), 4.26 (m, 5H), 6.83 (d, 1H,
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10.1002/ejoc.201801490
European Journal of Organic Chemistry
FULL PAPER
J = 7.7 Hz), 7.27 (t, 1H, J = 5.6 Hz), 7.32 (t, 2H, J = 7.4 Hz), 7.41 (t, 2H, J
= 7.4 Hz), 7.67 (d, 2H, J = 7.4 Hz), 7.88 (d, 2H, J = 7.4 Hz) 8.01 (t, 1H, J
= 5.4 Hz), 8.35 (s, 1H), 8.44 (s, 1H), 8.97 (bs, 2H); 13C NMR (DMSO-d6,
75 MHz): δ ppm 26.1, 28.2, 29.0, 38.4, 39.9, 43.4, 46.8, 54.2, 65.2, 78.0,
118.0, 120.1, 125.1, 127.0, 127.6, 140.7, 143.8, 143.9, 145.7, 148.9,
150.9, 155.4, 156.1, 172.6; HPLC, RT= 1.54 min; MS (ESI+): m/z calcd
for C32H39N8O5 615.3 [M+H]+, found 615.2.
experiments. Several silver colloid batches were synthesised using the
classical Creighton’ method[22] and analyzed as described in Soualmia et
al..[13] For optimal reproducibility of SERS experiments, the samples were
inserted in glass tubes using the following order: 80 µL of colloid, 10 µL
of salt and 10 µL of tested aqueous solution. The SERS spectra of these
latter were immediately recorded.
Monitoring of the trypsin activity by HPLC
Synthesis of compounds 22
The reaction mixture (1.2 mL) contained 100 mM TRIS-HCl and 10 mM
CaCl2, pH 8.0, 37°C, with 22b or Boc-VPR-AMC varying from 37 to 150
µM. Reactions were initiated by the addition of trypsin (final concentration
0.2 µM). Aliquots of 100 µL were periodically removed from the reaction
mixture and trypsin was inactivated by the addition of 2 µL of 2 N HCl to
each aliquot. 10 µL of the mixture was injected and the quantity of Boc-
VPR-OH released at various times was quantified by HPLC (λ= 214 nm).
- Boc deprotection: removal of the Boc protecting group was carried out
using a solution of TFA/DCM 1/1 v/v at room temperature for 1h. The
solution was then evaporated to dryness under vacuo and the residue
was used in the next step without purification.
-
Peptide coupling conditions: after deprotection, the residue was
dissolved in DMF (5 mL), basified with triethylamine. 1-
Hydroxybenzotriazole monohydrate (1.1 eq), EDCI (1.1 eq) and Boc-AA-
OH (1 eq) were added. The solution was stirred at room temperature for
2 h and then evaporated to dryness. The residue was dissolved in 25 mL
of ethyl acetate and washed with a saturated NaHCO3 solution (2 x 25
mL) and then with brine (1 x 25 mL). The organic layer was dried over
Na2SO4, filtered, and the solvent was concentrated under vacuum.
Acknowledgements
This work was supported by Université de Montpellier, Institut
National pour la Recherche Médicale (INSERM), Université
Pierre et Marie Curie (UPMC), Centre National de la Recherche
Scientifique (CNRS). The authors are grateful to the French
Ministry of Research and Education for F. S. PhD fellowship.
- Cbz deprotection: the Cbz-protected derivative was dissolved in ethanol
(20 mL) and 10% activated palladium on carbon was added. Hydrogen
gas was bubbled into the solution at 25°C until completion of the reaction
(HPLC monitoring). Then, the solution was filtered on a pad of celite.
Deprotected derivative was obtained after evaporation to dryness under
vacuum.
Keywords: Serine protease • Probe • Adenine • Surface
Enhanced Raman Spectroscopy • Peptide
[1]
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for 1h. The solution was then evaporated to dryness under vacuum and
the residue was used in the next step without purification.
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21a in acidic conditions, the crude was dissolved in 10 mL of DCM. Then,
were added triethylamine until alkaline pH (~9) and then 1.1 eq of
succinic anhydride. The solution was stirred at room temperature until
completion (HPLC monitoring). The solution was evaporated to dryness
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preparative HPLC.
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93, 202–213.
- Guanidinium formation: to a solution of crude compound 22c in 10 mL
of DMF were added triethylamine until alkaline pH (~9) and then 3 eq. of
pyrrazole-1-carboxamide. The solution was stirred at room temperature
until completion (HPLC monitoring). The solution was evaporated to
dryness under vacuum and the residue was washed with EtOAc and then
with Et2O. The crude was dissolved in DMSO (~2 mL) and then purified
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Compound 22a (Suc-Leu-Leu-Val-Tyr-CH2-CH2-Adenine). HPLC, RT=
1.54 min; MS (ESI+) m/z: calcd for C37H55N10O8 [M+H]+ 767.0, found
767.3
[12] I. A. Larmour, K. Faulds, D. Graham, Chem. Sci. 2010, 1, 151–160.
[13] F. Soualmia, S. A. Touhar, L. Guo, Q. Xu, M. V. Garland, P. Colomban,
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[14] A. Grondin, D. C. Robson, W. E. Smith, D. Graham, J. Chem. Soc.
Perkin Trans. 2 2001, 2136–2141.
Compound 22b (Boc-Val-Pro-Arg-CH2-CH2-Adenine). HPLC, RT= 1.03
min; MS (ESI+) m/z: calcd for C28H47N12O5 [M+H]+ 631.4, found 631.3
[15] L. Grajcar, C. El Amri, M. Ghomi, S. Fermandjian, V. Huteau, R.
Mandel, S. Lecomte, M.-H. Baron, Biopolymers 2006, 82, 6–28.
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384–390.
SERS evaluation
The Raman spectra were recorded on
a Senterra Bruker Optics
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instrument (Ettlingen, Germany) using a 532nm diode laser source and a
1200 lines/mm grating (resolution < 4 cm-1). The Rayleigh filtering is
achieved using Notch Filters. The laser power was generally 20 mW, the
acquisition time was 20s (up to 10 accumulations). The spectra of the
various samples were recorded in at least three independent
[20] G. B. Chheda, R. H. Hall, Biochemistry (Mosc.) 1966, 5, 2082–2091.
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[21] N. Narayanaswamy, M. B. Avinash, T. Govindaraju, New J. Chem.
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European Journal of Organic Chemistry
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Title
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SERS Probes*
Nicolas Masurier,* Feryel Soualmia,
Pierre Sanchez, Valérie Lefort, Mia
Roué, Ludovic T. Maillard, Gilles Subra,
Aline Percot] and Chahrazade El Amri*
Page No. – Page No.
We report an efficient way to synthesize challenging peptide-adenine conjugates,
as alternative to the aminomethylcoumarine (AMC)-serine proteases substrates.
Synthesis of peptide-adenine
conjugates as a new tool for the
protease activity monitoring
*one or two words that highlight the emphasis of the paper or the field of the study
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